Myoglobin strikes back

Abstract

Over the last half century, myoglobin (Mb) has been an excellent model system to test a number of concepts, theories, and new experimental methods that proved valuable to investigate protein structure, function, evolution, and dynamics. Mb's function, most often considered just an oxygen repository, has considerably diversified over the last 15 years, especially because it was shown to have a role in the biochemistry of quenching and synthesizing nitric oxide in the red muscle, thereby protecting the cell. To tackle protein's structural dynamics by innovative biophysical methods, Mb has been the best prototype; laser flash technology made it possible to obtain molecular movies by time-resolved Laue crystallography (with ps resolution). This approach unveiled the complexity of the energy landscape and the structural basis of the stretched interconversion between conformational substates of a protein.

Figure 1

Simplified kinetic scheme depicting events following photolysis of liganded Mb (A). The photolysed ligand (O₂, CO, NO, and others) populates the so-called distal pocket (primary docking site, B), and thereafter may: (i) diffuse out into the solvent (S) via the His-gate, the most probable path according to Scott et al.; (ii) rebind to the metal by an intramolecular geminate process B → A; (iii) diffuse into the matrix secondary docking sites (C), following a path traced by the Xe binding cavities (as shown by time-resolved Laue crystallography). Escape out into the solvent is occurring also via other pathways over-and-above the His-gate.,

Figure 2

Structural changes in the heme vicinity for the mutant called YQR-Mb, from 100 ps to 316 ns after CO photolysis. YQR-Mb is a triple mutant (L29Y/H64Q/T67R) used for this experimental session because of favorable properties (zero CO geminate recombination, slow bimolecular rebinding, and excellent sturdy crystals). (F_light − F_dark), the difference electron density maps, are in red for negative and in green for positive (contoured at 3.0 σ); these are overlaid on models of YQR-MbCO (in yellow) and YQR-Mb (in blue). It may be seen that at 100 ps, the negative density (red) of the Fe²⁺CO on the distal side of the heme is very prominent; other detectable changes are the initial distortion of the heme and motions of the distal Tyr29 and Gln64. These structural changes continue to grow with time and are fully developed in the frame at 316 ns. In all the frames, a blue bar indicates the position of photolyzed CO migrating initially to the Xe4 pocket (distal, at the top in the 100 ps frame) and later to the Xe1 pocket (proximal, at the bottom, better seen in the frame at 316 ns) (from Bourgeois et al., modified).

Figure 3

Time dependence of difference electron densities for key structural features, after photolysis of YQR-MbCO. Numerical values represent the integral of the positive electron density beyond 3.0 σ, corrected for variations in photolysis yield and normalized to the negative bound CO feature (arbitrary value of 1). (a) Key features that appear promptly: the density of the Fe popping out of the heme plane without delay remains time independent; while distortion of the heme (see Fig. 2) and motion of Tyr29 follow an heterogeneous time course being already detectable in the first frame (density of a 0.10–0.15 at 0.1 ns) but increasing with time. (b) Amino acid residues involved in the strain of the CD turn clearly lag behind the conformational changes of the heme and of Tyr29 on the distal side (see above). (c) Population of CO in the Xe1 and Xe4 pockets, and conformational changes of the E-helix: CO appears promptly after photolysis in the Xe4 pocket, on the distal side of the heme (with 0.035–0.045 density), and begins to diffuse inside and populate the Xe1 pocket on the proximal side starting from ∼5 ns. The migration of CO is approximately synchronous with larger scale conformational changes of the E-helix, suggesting that long-scale intramolecular diffusion demands more substantial structural changes of the globin. At much longer times (e.g., toward ms) CO rebinds largely via the opening of the His-gate, as shown by Scott et al. studying many different mutants of sperm whale Mb (from Bourgeois et al., modified).